Simultaneous enantioselective determination of seven psychoactive drugs enantiomers in multi-specie animal tissues with chiral liquid chromatography coupled with tandem mass spectrometry

Article abstract of DOI:10.1016/j.foodchem.2019.125241

In stock farming, illegal use of antipsychotics has caused the food safety problem. This paper presents for the first time, a multi-residues method for the simultaneous enantioselective determination of seven antipsychotics in pork, beef and lamb muscles. The behaviors of Chiralpak AGP toward changes in pH and organic modifier in mobile phase were studied, and all analytes were rapidly separated within 30 min. The calibration curves of all enantiomers were linear in the range of 1–250 ng g^?1, with correlation coefficient above 0.9931. The recoveries of the targeted compounds were higher than 82.1%, with repeatability and intermediate precision lower than 18.2% and 17.4%, respectively. In three matrices, the limit of detection and limit of quantification ranged from 0.20 to 0.65 ng g^?1 and from 0.40 to 1.00 ng g^?1, respectively. The proposed method can be used to provide additional information for the reliable risk assessment of chiral antipsychotics.

Full text of DOI:10.1016/j.foodchem.2019.125241

Accepted Manuscript
Simultaneous enantioselective determination of seven psychoactive drugs enan-
tiomers in multi-specie animal tissues with chiral liquid chromatography cou-
pled with tandem mass spectrometry
Bolin Zhu, Shuang Li, Li Zhou, Qing Li, Xingjie Guo
PII:
S0308-8146(19)31349-4
https://doi.org/10.1016/j.foodchem.2019.125241
125241
DOI:
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Reference:
FOCH 125241
Food Chemistry
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11 April 2019
22 July 2019
Please cite this article as: Zhu, B., Li, S., Zhou, L., Li, Q., Guo, X., Simultaneous enantioselective determination of
seven psychoactive drugs enantiomers in multi-specie animal tissues with chiral liquid chromatography coupled
with tandem mass spectrometry, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem.2019.125241
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Simultaneous enantioselective determination of seven psychoactive drugs enantiomers in
multi-specie animal tissues with chiral liquid chromatography coupled with tandem
mass spectrometry
Bolin Zhu, Shuang Li, Li Zhou, Qing Li*, Xingjie Guo*
Affiliation: School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road Shenhe District,
110016 Shenyang, Liaoning Province, P. R. China
*Correspondence: Professor Xingjie Guo; Professor Qing Li, School of Pharmacy, Shenyang Pharmaceutical
University, 103 Wenhua Road Shenhe District, 110016, Shenyang, Liaoning Province, P. R. China.
E-mail: syykdx410@163.com (Xingjie Guo); lqyxm@hotmail.com (Qing Li).
Tel./Fax.: 86-24 2398 6289
1

Abstract
In stock farming, illegal use of antipsychotics has caused the food safety problem. This paper presents for
the first time, a multi-residues method for the simultaneous enantioselective determination of seven
antipsychotics in pork, beef and lamb muscles. The behaviors of Chiralpak AGP toward changes in pH and
organic modifier in mobile phase were studied, and all analytes were rapidly separated within 30 min. The
calibration curves of all enantiomers were linear in the range of 1-250 ng g-1, with correlation coefficient above
0.9931. The recoveries of the targeted compounds were higher than 82.1%, with repeatability and intermediate
precision lower than 18.2% and 17.4%, respectively. In three matrices, the limit of detection and limit of
quantification ranged from 0.20 to 0.65 ng g-1 and from 0.40 to 1.00 ng g-1, respectively. The proposed method
can be used to provide additional information for the reliable risk assessment of chiral antipsychotics.
Keywords: Antipsychotics; Chiral separation; Enantiomeric determination; Meat productions.
2

1. Introduction
Psychotropic drugs are illegally applied on a large scale for different purposes in modern farming. For
example, antipsychotics are often used in animal husbandry to enhance the feed conversion ratio by reducing
animal activity. In addition, antipsychotics are administered to farm animals (such as pig, bovine and lamb) to
reduce stress during their transport to slaughterhouse. Such stress will cause the loss of meat quality and even
premature death. Clinical diagnosis and forensic toxicological cases have shown that antipsychotics in animal
can lead to overdose dependence, excessive sedation, anesthesia, coma, and even death (Wang, Zhao, Qiu, &
Zhou, 2006). Illegal administration of drugs before slaughter gives rise to residues in edible animal tissues
because these antipsychotics are often used just a few hours before slaughter. The accumulation of
antipsychotics in animal meat products may enter the human body via food chain. As a result, these
antipsychotics residues will cause a range of side effects, including hypotension, lethargy, nightmares or
hyperprolactinaemia, and thus bring a potential risk to consumers. Previous study proposed that the psychotropic
drugs had potential genotoxic activity (Gocke, 1996). The use of phenothiazine derivatives (chlorpromazine and
propionylpromazine) is totally prohibited. The joint FAO/WHO Expert Committee on Food Additives (JECFA)
showed concern about the safety of drug residues including antipsychotics in food in 1987, but these
antipsychotics are still illegally used. Thus, from the food safety point of view, quantification and confirmation
of antipsychotics residues in animal productions are necessary for human health and fair competition.
In the current market, various classes of antipsychotics have come into use, such as selective serotonin
reuptake inhibitors (SSRIs), serotonin and norepinephrine reuptake inhibitors, noradrenergic and specific
serotonergic antidepressants, etc. Herein, seven chiral antipsychotics belonging to different families are chosen
as the targeted analytes, including cyamemazine, promethazine, fluoxetine, oxazepam, mirtazapine, bupropion
and phencynonate. The chemical structures of seven antipsychotics are shown in Fig. 1. As seen, all the targeted
3

seven antipsychotics contain heteroatoms in their structures. The synthesis and biological importance of different
heteroatom-containing compounds have become the hot topics in recent years (Ahankar, Ramazani, lepokura,
Lis, & Joo, 2016; Dayyani, Khoee, & Ramazani, 2015; Garmroodi et al., 2016; Jalili-Baleh et al., 2018; Lauro et
al., 2019; Malekzadeh, Ramazani, Tabatabaei-Rezaei, & Niknejad, 2017; Shalbafan, Behbehani & Ghasemzadeh,
2019; Yavari, & Ramazani, 1997). For example, doxorubicin has shown great treatment potential, as it possesses
the ability to combat rapidly dividing cells and slow disease progression (Shalbafan et al., 2019). Lauro et al.
(2019) synthesize a diaza-bicyclo-naphthalen-oxiranyl-methanone derivative using some chemical tools, which
could change the biological activity of cytochrome P450-17A1 enzyme and thus be translated as good candidates
for prostate cancer. The natural and synthesized chroman-4-one-based structures would be used asmulti-target-
directed ligands (MTDLs) for Alzheimer's disease (Jalili-Baleh et al., 2018). For the seven heteroatom-
containing antipsychotics, the quantitative determination of their contents in meat products is of great significant,
to obtain more information about the toxicity and biological behaviors.
Stereo-chemistry in drugs has gained more and more attention because two enantiomers always show
different pharmacokinetic profiles and biological activities. The use of racemic drugs may reduce curative effect
and cause adverse effect owing to the presence of either inactive or toxic enantiomer. Lot of the used
antipsychotics contain one or more chiral centers and until now they are administrated as racemates (Nageswara-
Rao, & Guru-Prasad, 2015). Metabolism of fluoxetine was found to be enantioselective in human, with the (R)-
fluoxetine being metabolized faster than the (S)-enantiomer. Toxicity tests of fluoxetine also proved strong
enantiomer-dependent (Andrés-Costa et al., 2017). Recent finding shows that promethazine displays
stereoselectivity in its pharmacokinetics, and thus two enantiomers exhibit different biological profiles toward
attractive molecular targets including cytokines (Borowiecki, 2015). There are evidences that (+/-)-mirtazapine,
(R)-(-)-mirtazapine and (S)-(+)-mirtazapine possess different pharmacokinetic and pharmacodynamic properties.
4

(S)-(+)-mirtazapine is a more potent receptor antagonist than its antipode at nearly all binding sites, except for 5-
HT3 and the muscarinic receptors (M1–M5) (Freynhagen, Vogt, Lipfert, & Muth-Selbacha, 2006). Trough plasma
level of (R)-(-)-mirtazapine is approximately 2-3 times higher than that of the (S)-(+)-isomer, which would be
attributed to the strong preference of CYP2D6 for the (S)-(+)-isomer (Dodd, Burrows, & Norman, 2000). In the
case of bupropion, the pharmacological activities of two enantiomers and its metabolites are reported to be
significantly different. Hydroxylation of (S)-(+)-bupropion by recombinant CYP2B6 is found to be 3-fold
greater than (R)-(-)-bupropion and the same by human liver microsomes is found to be 1.5-fold greater than the
(R)-(-)-isomer (Coles, & Kharasch, 2008). As is well known that pork, beef and lamb are the main meat wares in
our daily live, residues of antipsychotics enantiomers, especially the highly active, toxic or even dangerous
stereoisomer, in these products will be harmful to consumers. The potency and toxicity relative to the
enantiomers of antipsychotics have not been fully characterized. With respect to these factors, enantioselective
determination of chiral antipsychotics in animal food product is a very important branch of analytical chemistry,
to provide information about chemical composition and quality control, to ensure compliance with food and
trade laws, also to assure product authenticity and safety. Up to now, there is a lack of literature working on the
enantioselective determination of antipsychotics in meat samples.
HPLC has become the most commonly used technique for analytical chemistry, especially in the
pharmaceutical and biomedical fields (Ganni, Kumar, Jain, Kumar, Shrivastava, & Kumar, 2019; Rezaei, &
Ramazani, 2019; Rezaei, Ramazani, & Hokmabadi, 2018; Rezaei, Ramazani, & Rouhani, 2017; Vyas, Nathwani,
Patel, Patel, & Patel, 2019;). Recently, more sensitive and specific mass spectrometry techniques hyphenated to
gas and liquid chromatography are widely applied for screening and confirmatory analysis. In cases of the seven
tested analytes, a high number of GC-MS and LC-MS/MS methods are developed in order to quantify racemates
or single enantiomers of antipsychotics in human matrices (including plasma, serum, urine and oral fluid). For
5

instance, Kirchherr, and Kü hnVelten (2006) have established a method for the quantitative determination of
forty-eight antidepressants and antipsychotics in human serum by HPLC-MS/MS. da Fonseca, Moreno, Barroso,
Costa, Queiroz and Gallardo (2013) developed a GC-MS/MS method for the determination of seven selected
antipsychotic drugs in human plasma. LC-MS method for the enantioselective analysis of mirtazapine, dimethyl
mirtazapine and 8-hydroxy mirtazapine in human plasma and urine has previously reported (De Santana, Jabor,
Cesarino, Lanchote, & Bonato, 2010). The GC-MS/MS method for the determination of seven antipsychotics in
plasma and oral fluid samples has been developed by Rosado et al. (2018). Based on the previous literatures, the
antipsychotic residues are mostly detected in human plasma, urine, or serum samples, but relatively less attention
has been paid to the antipsychotic residues in food, such as pork, beef and lamb. Only few reports on the
determination of racemic antipsychotics in animal tissues have been published. For example, assay based on GC-
MS has been used to detect four antipsychotics residues in swine muscle and liver tissues (Cheng, Zhang, Shen,
Wu, & Zhang, 2010). He, Wang, Zhang, Liu and Fang (2012) reported a LC-MS/MS method for the
simultaneous determination of the tranquilizers (chlorpromazine, promethazine, diazepam, azaperone, and its
metabolite, azaperol) and a β-blocker (carazolol) in edible swine tissues. Cheng, Shen, Zhang, Zhang and Zhang
(2015) presented a rapid method for the determination of three tranquillizers (diazepam, chlorpromazine, and
promethazine) in lamb liver by UPLC-MS/MS. For the seven targeted antipsychotics, there have been few
reports on the determination of racemic promethazine and fluoxetine in animal tissues. The quantification of
other five antipsychotics in animal tissues has not been reported, which leads to lack of data for risk assessment.
Furthermore, no study has reported on the enantioselective determination of chiral antipsychotics in animal
tissue. Therefore, it is vital to develop the enantioseparation and enantioselective determination method for
antipsychotics in animal products to provide more results for evaluating the biotoxicity and monitoring food
safety.
6

Based on this, in the present study, we first separated the enantiomers of seven chiral antipsychotics using
the ɑ1-acid glycoprotein (AGP) chiral column, and then a reverse phase UPLC-MS/MS method was optimized,
validated and applied for the enantiomeric determination of seven antipsychotics in pork, beef and lamb meats.
The different parameters affecting chiral separation and extraction recovery were studied. The method was
demonstrated to be sensitive, selective and easily applicable to analyze animal tissue samples, and potentially
beneficial for research in this area. To date, this is the first report for the simultaneous enantioselective analysis
of seven antipsychotics in multi-specie animal tissues (pork, beef and lamb meats).
2. Materials and Methods
2.1 Chemicals and Reagents
Seven antipsychotics standards: R/S(±)-cyamemazine, R/S(±)-promethazine, R/S(±)-fluoxetine, R/S(±)-
oxazepam, R/S(±)-mirtazapine, R/S(±)-bupropion and R/S(±)-phencynonate were purchased from China
Standard Material Centers (Beijing, China). All standards were of >98.5% purity. The chemical structures of the
seven psychoactive drugs are shown in Fig. 1. MS-grade ammonium acetate, acetic acid, methanol and
acetonitrile were provided by Sigma-Aldrich (Beijing, China). All other reagents were of analytical grade and
purchased from Yuwang Technology (Shandong, China). Ultrapure water was obtained from Jilin Wahaha
Foods Co., Ltd. (Jilin, China) and was used in all the experiments. The Cleanert C18 solid-phase extraction
columns (1000 mg, 6cc) were obtained from Agela Technologies (Tianjin, China). Stock standard solutions for
the seven antipsychotics (1 mg mL-1) were prepared in methanol and stored at -20 ◦C. Working mixture solution
(10 μg mL-1) was prepared by combining aliquots of stock solutions followed by subsequent dilution with
methanol. The spiked solutions were then made immediately before the analysis by serially diluting the working
mixture solution with methanol. Accordingly, matrix-fortified standard calibration curves at seven concentration
levels (1, 5, 10, 50, 100, 200 and 250 ng g-1 of each enantiomer) were made by spiking certain amounts of
7

standards to the blank matrices.
2.2 Sample preparation
The pork, beef and lamb muscle tissue samples were bought from the local market in Benxi (China). These
samples were homogenized and stored at -20 ◦C until use. These samples were prescreened to confirm that they
did not contain the targeted antipsychotics and thus used for the method validation. Each aliquot (5.00 ± 0.01 g)
of homogenized sample (pork, beef and lamb muscle tissues) was weighed and transferred into a 50 mL
polypropylene centrifuge tube. About 5 g of anhydrous sodium sulfate was added until free-flowing powder
could be obtained after vortex-mixing. Acetonitrile (15 mL) was also added to the mixture, which was vortex-
mixed for 2 min. The sample was then placed in an ultrasonic bath for 20 min at 40 ◦C. After cooling to room
temperature, the mixture was centrifuged at 4000 rpm for 10 min to form distinct liquid layer from the liquid and
solid mixture. The supernatant was transferred into a 100 mL evaporation flask and then concentrated to dryness
by a rotary evaporator at 60 ◦C. Residue was re-dissolved with 20 mL methanol-water (5:95, v/v) for further
purification.
2.3 Sample purification
The Cleanert C18 solid-phase extraction (1000 mg, 6 cc) column was preconditioned with 6 mL of
methanol and then equilibrated with 6 mL of water. Then, the 20 mL sample solution was loaded onto the SPE
column. When the sample had eluted under gravity, the column was washed with deionised water (6 mL).
Vacuum was applied to the cartridge for 2 min to completely dry the resin. Finally, elution was carried out with
6 mL of methanol, and the elute was evaporated to dryness under a gentle stream of nitrogen at 50 ◦C. The
obtained residue was reconstituted with 0.2 mL of methanol-water (50:50, v/v) and filtered through a 0.22 μm
syringe filter before LC-MS/MS analysis.
2.4 LC-MS/MS
8

All samples were analyzed using chiral liquid chromatography performed on Waters AcquityTM UPLC
system (Waters Corp., Milford, MA, USA) composed of AcquityTM UPLC binary pump solvent management
system, an AcquityTM UPLC autosampler and a thermostatic column compartment. The simultaneous
enantioseparation of the seven psychoactive drugs was achieved on a Chiralpak AGP column (150 mm×4.6 mm,
I.D. 5 μm), coupled to a Chiralpak AGP guard column (10 mm×4.6 mm, I.D. 5 μm), which were supplied by
Daicel Chiral Technologies Co., Ltd. (Tokyo, Japan). Chiralpak AGP was protein-based chromatographic
support with ɑ1-acid glycoprotein as the chiral selector. A gradient mode was used to separate and elute targeted
analytes from the column. The optimized enantiomeric separation was obtained with the mobile phase consisting
of methanol (solution A) and 10 mM ammonium acetate buffer adjusted at pH 4.5 (solution B), with a flow rate
of 0.6 mL min−1. The initial mobile phase was 100% solution B. From 0 to 10 min, the percentage of solution A
was increased to 15% linearly and held from 10 to 30 min. The mobile phase returned to the initial composition
and equilibrated for 10 min before the next injection. Column temperature and autosampler temperature were set
at 30 °C and 4 °C, respectively. The injection volume was 10 μL.
Mass spectrometer was carried out on a Waters Micromass QuattromicroTM API mass spectrometer (Waters,
Milford, USA) equipped with an electrospray ionization source. All data collected was processed using
MassLynx 4.1 software (Waters Corp., Milford, MA, USA). Analysis was performed in positive ion mode. The
ionization source conditions were as follows: capillary voltage of 3.0 kV and source temperature of 150 °C.
Nitrogen gas was used as the desolvation gas and set to a flow rate of 16666.67 mL min-1 with a temperature of
500 °C. The collision gas by ultra-high purity argon was held at 0.12 mL min− 1. The mass spectrometer was
operated in product ion scan mode to select the transition, and in multiple reaction monitoring (MRM) mode to
quantify the analytes in samples. The MRM transition of each antipsychotic was optimized by the direct infusion
of individual standard solution at a concentration of 100 ng mL-1. Two MRM transitions were monitored for
each analyte. The most sensitive product ion was selected for quantification, and the other transition was used for
confirmation. Optimized MRM transition, collision energy and capillary voltage are depicted in Table 1.
3. Results and Discussion
9

3.1 Optimization of LC-MS/MS
3.1.1 Column selection
The selection of column is important in separation of chiral antipsychotics. In the preliminary experiments,
we initially screened two polysaccharide-based chiral stationary phases (CSPs), namely tris-(4-methylbenzoate)
of cellulose (Chiralcel OJ-RH) , tris-(3-chlorophenylcarbamate) of amylose (Chiralpak ID), to resolve the seven
antipsychotics. Both columns were performed under reversed phase mode. With acetonitrile/water as the mobile
phase, Chiralpak ID showed enantioselectivity for oxazepam (Rs=0.9), while no separation was observed for
other analytes. On Chiralcel OJ-RH, two analytes of promethazine (Rs=2.42) and phencynonate (Rs=2.44)
obtained complete separation, with acetonitrile/water/DEA (60/40/0.1, v/v/v) as the mobile phase (data not
shown). The enantiomers of other five analytes were not separated on Chiralcel OJ-RH. Based on the above
results, polysaccharide-typed CSPs were not capable of the simultaneous enantioseparation of the targeted
antipsychotics. Due to the broad application range and LC-MS compatibility of protein-based CSPs, in particular
α1-acid glycoprotein, Chiralpak AGP column was also attempted for the separation experiments. As a result,
Chiralpak AGP proved to be the most efficient column, on which all analytes were separated into individual
enantiomer. Chiralpak AGP was thereby selected as the optimal CSP to further optimize the separation.
3.1.2 Mobile phase optimization
The enantioseparation performance of Chiralpak AGP may be influenced by several parameters, such as the
buffer type and its concentration, the pH value of the buffer, the organic modifier type and its content
(Michishita, Franco, & Zhang, 2010). The mobile phase optimization on Chiralpak AGP requires adjustment of
these parameters. On the basis of all published studies on Chiralpak AGP, ammonium acetate buffer is generally
applied on Chiralpak AGP because of its good buffering ability and LC-MS compatibility. The pH of 5.8 is the
mean value of the recommended pH value (4.5-7.0) for Chiralpak AGP, and IPA is reported to be the most
10

versatile modifier. Thus, 10 mM ammonium acetate buffer adjusted at pH 5.8 admixed with 5% IPA was chosen
as the initial mobile phase to start the enantioseparation (Table 2). As a consequence, five out of the seven
analytes (except for mirtazapine and bupropion) were baseline-resolved along with too longer retention (over 40
min). In most cases, antipsychotics were found to be highly retained on Chiralpak AGP column. It has been
reported that buffer pH is the most vital parameter to regulate the retention (Michishita et al., 2010). In acidic
mobile phase, the chiral selector on Chiralpak AGP is negatively charged, which can provide ionic interaction
for these tested antipsychotics (basic compounds) (Michishita et al., 2010). Thus, lower pH value is expected to
decrease the retention time, and pH 4.5 was chosen in our present study. Considering that two analytes of
mirtazapine and bupropion were not separated with IPA/10 mM ammonium acetate buffer as the mobile phase,
other organic modifiers were subsequently tested to achieve more successful enantioresolution.
The use of acetonitrile as organic modifier was subsequently studied on Chiralpak AGP. Mobile phase
containing different percentage of acetonitrile and 10 mM ammonium acetate buffer (pH=4.5) was tried, and six
analytes (except for bupropion) obtained the satisfactory separation (Table 2). In the case of bupropion, the poor
resolution was probably due to the fast elution (k’<0.7), and the complete removal of acetonitrile from the
mobile phase allowed an increase in the retention and, more importantly, an improvement of the resolution up to
1.59. Besides acetonitrile, methanol is the other alternative organic modifier on Chiralpak AGP. Using methanol
instead of acetonitrile as the modifier, significant decrease in analyte retention (k1) and improvement in
enantioselectivity (α) were observed for all analytes (Table 2). Based on the results obtained with three modifiers,
it could be concluded that methanol was more suitable for enantioseparation of targeted antipsychotics because
of the comprehensive separation ability, higher enantioselectivity as well as shorter retention.
It should be noted that the chromatographic behaviors of enantiomers were dependent on the properties of
compounds and the composition of mobile phase. The enantiomers of mirtazapine and bupropion were weakly
11

retained on Chiralpak AGP compared with other analytes. The adjustment of methanol content to a lower value
(~5%) would regulate their retention and guarantee the baseline separation. But for other five analytes, the
analysis time increased significantly and their peak shapes broadened when lower methanol content was added to
mobile phase. For the purpose of simultaneous enantioseparation, gradient elution was necessary, which began
with 100% ammonium acetate buffer (10 mM, pH 4.5), increased linearly in 10 min to 15% of methanol, and
then maintained this proportion for 20 min. Under the optimized mobile phase, baseline resolutions (Rs > 1.5)
were achieved for all analytes within half an hour. MRM chromatograms of the spiked pork samples (10 ng g-1
for each enantiomer) are presented in Fig. 2 (b).
3.2 Sample preparation
3.2.1 Extraction
Sample preparation is the crucial step in the analysis of complicated biological matrix, which is highly
influenced by the physical and chemical properties of targeted analytes. The main purposes of sample
preparation are to concentrate analytes in matrix, to remove interferences from complicated matrix and also to
prepare analytes in suitable form for the following chromatographic analysis. According to the previous papers,
the extraction of antipsychotics from meat matrices was always accomplished with organic solvent extraction
and SPE. The selection of appropriate extraction solvent and sorbent is highly related to the properties of the
studied analytes. However, no paper reports the simultaneous extraction of the seven targeted antipsychotics
from meat matrices. Based on this, the pretreatment procedure in our study was established by the modification
of published literatures. Prior to determination by LC-MS/MS, the main parameters that affected the extraction
efficiency such as the type of the extraction solvent, extraction times, the type of SPE cartridge, and the content
of loading buffer were carefully studied.
Previously, experiments to extract antipsychotics from animal tissues were frequently performed with
12

acetonitrile and ethyl acetate (Cheng et al., 2010; He et al., 2012; Cheng et al., 2015; Zhang, Shao, Yin, Wu, &
Duan, 2009; Zhang et al., 2014; Zhang et al., 2016). Cheng et al. (2015) extracted three antipsychotics (diazepam,
chlorpromazine and promethazine) from lamb live with ammonia in ethyl acetate. In the study of Zhang, Shao,
Yin, Wu, and Duan (2009), thirty targeted drugs (including 11 antipsychotics) were extracted with acetonitrile in
animal tissues (pork kidney, liver, muscle; bovine muscle). Zhang et al. (2016) extracted eight tranquilizers
residues including chlorpromazine, diazepam, nitrazepam, oxazepam etc in pork with acetonitrile as extraction
solvent. It has been reported that alkaline compounds tend to dissolve in no-polar solvent (ethyl acetate) as
molecular form in alkaline conditions, but dissolved in polar solvent (acetonitrile) as ionic form under acidic
condition (Cheng et al., 2015). On the basis of the published literatures, our present study evaluated the
extraction efficiencies of four kinds of extractants, including acetonitrile, formic acid-acetonitrile (1:99, v/v),
ethyl acetate, 25% of ammonium hydroxide-ethyl acetate (10:90, v/v). As depicted in Fig. 3 (a), the use of ethyl
acetate as the extractant led to the relatively low recoveries of targeted analytes. Moreover, it was found that the
extracts obtained with ethyl acetate showed muddy colors, thus increasing the matrix effect for most analytes.
The addition of 25% ammonium hydroxide to ethyl acetate did not improve the recoveries. In comparison, the
use of acetonitrile as the extractant provided superior extraction efficiencies for seven analytes with recoveries
higher than 78% (Fig. 3 (a)). This phenomenon is inconsistent with the report of Cheng et al. (2015). It could be
explained by the different properties of different antipsychotics. The seven analytes possess a wide range of
polarity, with logarithms of oil-water partition coefficient (log P) for the seven analytes from 2.71 (bupropion) to
4.27 (fluoxetine), and thus a polar organic solvent of acetonitrile is more workable. Considering that the seven
analytes were alkaline, formic acid-acetonitrile (1:99, v/v) was then attempted, leading to poor extraction of
cyamemazine, promethazine and phencynonate (recoveries<5%). Therefore, acetonitrile was selected as the
optimum extractant for the seven analytes. We tried to repeat the solvent extraction twice to improve the
13

recoveries, but no improvement in recovery was observed. Thus, the one-step extraction with acetonitrile as the
extractant was chosen for present study. In addition, anhydrous sodium sulfate was adopted to dry the samples
and also improved the extraction efficiency of hydrophobic analyte.
3.2.2 Purification
Liquid-liquid extraction (LLE) and SPE are often used for the extraction and purification of antipsychotics
from biological matrices. However, LLE always leads to mutual solubility and emulsion formation of analytes in
two phases. For this reason, we subsequently tried to select appropriate SPE cartridges, after rotary evaporator
drying and redissolving with methanol-water. The cartridges recovery experiments were performed on three SPE
cartridges, including Cleanert C18 (1000 mg, 6 cc), PEP-2 (60 mg, 3cc) and NH2 (500 mg, 6cc). Results are
compared in Fig. 3(b). Through the further purification on PEP-2 cartridge, it was found that the PEP-2 was
unable to absorb bupropion in the extraction solvent. For the NH2 cartridge, poor recoveries (<20%) were
observed for fluoxetine, mirtazapine, bupropion and phencynonate. However, C18 cartridge would provide
higher recoveries for all the analytes. C18 is cheaper than other cartridges, so it was selected for the purification.
For the reverse-phase C18 cartridge, water-methanol was used as the loading solvent. Then, we also
optimized the ratio of methanol and water, and selected the ratio of water and methanol of 85: 15, 90: 10, 95:5,
98:2 (v/v). Fig. 3(c) shows the extraction recoveries for the seven analytes. As depicted, a decrease in extraction
recovery was observed with increasing the methanol content from 5 to 15%. Finally, the ratio of water-methanol
was determined as 95: 5 due to the highest recoveries for seven analytes.
Under above optimization of pretreatment procedure, a simple acetonitrile extraction step, followed by a
SPE cleanup using C18 cartridge was developed for the simultaneous extraction of seven antipsychotics from
multiple meat matrices. We acknowledge that the pretreatment method is established based on previous method.
Nevertheless, our research first report the extraction of cyamemazine, oxazepam, mirtazapine, bupropion and
14

phencynonate from multiple meat matrices. This procedure shows advantages compared with the published
literatures for antipsychotics analysis. In our study, a total amount of 15 mL acetonitrile is used for the extraction
step, which is solvent-saving than the methods developed by Cheng et al. (2015) and Zhang et al. (2009). In the
course of SPE, the C18 cartridge is more efficient, low-cost and easily-operated than other absorbents, such as
Oasis HLB (Zhang et al., 2016), NH2 (Zhang et al., 2009), MCX (Zhang et al., 2014), and Sep-pak VAC alumina
N (Cheng et al., 2015), leading to good cleanup and higher recoveries (>80%) for all the targeted analytes.
Nevertheless, the sample preparation with SPE procedure is time-consuming, which also encounters in other
literatures. Due to the complexity composition of meat matrices ( including protein and lipids), the use of SPE
for the cleanup is necessary.
3.3 Method and validation
The performance of the developed method was validated in terms of selectivity, linearity, limit of detection
(LOD), limit of quantification (LOQ), accuracy and precision, matrix effects (MEs), as well as the stability.
Blank muscle tissue samples were analyzed to verify the specificity of the method. Chromatograms of blank
sample and spiked sample are compared in Fig. 2. It was evident that no interfering specie was observed at the
retention of targeted analytes. Therefore, the method was proved to be selective for the determination of the
targeted antipsychotics in meat samples.
To prevent matrix suppression or sometimes enhancement effects, which may cause difference in absolute
response from the matrix compared to that of the standard, matrix-spiked samples were used as calibration
standards for evaluating response linearity. The matrix-fortified calibration curves were prepared using blank
meat samples spiked in the range of 1-250 ng g-1 at seven concentration levels. Each level was prepared in
triplicate. The spiked samples were extracted and cleaned up following the above-mentioned procedure.
Calibration lines were conducted from the peak areas of analytes (y-axis) against the nominal drug
15

concentrations (x-axis). Linear regression analysis was used to calculate the slope, intercept, and the correlation
coefficient (R2) of each calibration. The data calculated are listed in Table 3. It was clear that accepted linearities
(R2 > 0.9931) were achieved for all enantiomers in the range of 1-250 ng g-1, indicating a good correlation for
each targeted analyte.
The LOD was defined as the lowest concentration of analyte spiked in the meat sample which could be
detected with a signal-to-nosie (S/N) ratio of 3, while the LOQ was determined at the S/N of 10. The LODs and
LOQs of the present method for the antipsychotics enantiomers are shown in Table 3. LOQs of 0.40-1.00 ng g-1
and LODs of 0.20-0.65 ng g-1 were obtained.
The recovery experiments were carried out to evaluate the accuracy (expressed as recovery) and precision
(expressed as RSD). Five replicates of spiked samples at three different concentration levels (5, 10, 50 ng g-1 for
each enantiomer) were prepared on three different days. For recovery studies, 5.00 ± 0.01 g portion of meat was
placed in the container and then spiked with the standards, taken care to uniformly spread them on the sample.
Individual spiked sample was left for 30 min at room temperature to ensure the sufficient distribution in the meat
matrix, and then prepared according to the above preparation method. Method recovery was calculated from the
ratio of the mean values of the detected concentration (Cdet) and the nominal concentration (Cnom) as follows:
(Cdet/Cnom)×100%. Repeatability (or intra-day precision) was based on the RSD of the determined responses of
five replicated spiked samples at three concentrations on the same day. Intermediate precision (or inter-day
precision) was calculated using fifteen spiked samples (five replicates per concentration), which were analysed
one three consecutive days and also expressed as RSD. The within-laboratory reproducibility was obtained using
the fifteen spiked samples (five replicates at the lowest spiked concentration of 5 ng g-1 and analysed at three
occasions with three different operators). The results of average recovery and RSDs are presented in Table 4.
The method provided satisfactory average recoveries of 82.1-113.9% for all targeted enantiomer independently
16

of the matrix under study. The analytical method showed good repeatability from 2.0 to 18.2%, and also good
intermediate precision from 2.6 to 17.4%. The results of the recovery studies indicated that our method would
provide satisfactory accuracy and precision for the enantiomeric determination of antipsychotics in multi-specie
animal tissue samples.
In LC-MS/MS analysis, the response of analyte in biological matrix can be affected by interfering species
in the original biological sample, which is the matrix effects (MEs). This effect could result in signal suppression
or sometimes in enhancement of the analyte response. The peak areas for standards prepared in sample extracts
(post-extraction standard) divided by the corresponding peak areas of standards in methanol are defined as the
MEs. The post-extraction standards were prepared by extracting blank meat samples in according with the
procedure in Section 2.2 and 2.3. The final extracts were evaporated and re-dissolved in standards prepared in
methanol at appropriate concentrations. The matrix-suppression effect occurs when the value is lower than 100%,
and a ME value above 100% represents the matrix-enhancement. The MEs values of all analytes in three animal
tissues are listed in Table 3. The general suppression of signal in different meat matrices was observed.
Expecially, the MEs was more pronounced for bupropion, which was more polar than other analytes. It has been
reported that ionization efficiency of polar analyte is more affected by the coeluates of the matrix than that of
apolar analyte, because most of the coeluates of the matrix are polar (Zhao, Wang, Gao, Guo, & Zhao, 2019). To
compensate the signal suppression, matrix-matched standard curves were therefore adopted for the validation of
the analytical method and for the quantitative analysis of real samples.
The stability was checked by storing standard solutions, spiked samples and the extracts at 4 ◦C and -20 ◦C
at different periods. The mixed standard solutions (100 ng mL-1 of each enantiomer, prepared in methanol) of the
seven antipsychotics were stores at -20 ◦C for three months. The solutions were analyzed every month, and the
peak areas were compared with that obtained after preparation. The peak areas after storage were found to be
17

between 90% and 110% of the initial area. When stored at 4 ◦C, the mixed solution standards remained stable for
one month. Meanwhile, the spiked samples at the lowest fortification level of 1 ng g-1 and the extracts of the
spiked sample at 1 ng g-1 were stored at 4 ◦C and -20 ◦C after preparation, and measured immediately, 24h, 48h,
one week and two weeks later. The recoveries of the targeted analytes were within the accepted limit of ±15% of
the nominal concentration when stored at 4 ◦C for two weeks or at -20 ◦C for one month.
3.4 Application to real samples
Meat samples (a portion from the leg of slaughtered animals) were purchased from different markets of
Benxi (China). A total of thirty meat samples of pork, beef and lamb (ten of each) were determined. These
samples of about 100 g were preserved at 4 ◦C until arrival at laboratory. Then, each of the samples (5.00 ± 0.01
g portion) were homogenized and transferred to polypropylene centrifuge tube, labeled and stored in the -20 ◦C
freezer. After storage for about 3 hours, these samples were extracted and cleaned-up according to the above-
mentioned sample preparation and purification procedure, followed by the enantioselective determination of
enantiomers of seven antipsychotics by UPLC-MS/MS. Results showed that no antipsychotic was detected in
any of the animal tissues samples.
4 Conclusion
The present method, based on one-step acetonitrile extraction, SPE clean-up with Cleanert C18 cartridges
and chiral LC-MS/MS determination, is suitable for the enantioselective determination of seven antipsychotics
residues in pork, beef and lamb muscle tissue samples. With Chiralpak AGP as the chiral selector, chiral
separation of the seven antipsychotics was accomplished within half an hour. The sample preparation procedure,
including one-step acetonitrile extraction and C18 SPE, provided high extraction efficiencies for all the analytes.
Quantification at 1ng g-1 for the different kinds of meat samples by LC-MS/MS was readily achieved. In
conclusion, the developed method is fast, simple and sensitive, which can be advantageously adopted as the
18

routine procedure to monitor the concentration of antipsychotics enantiomers in the laboratories for food quality
and safety control because of its robustness and feasibility.
Acknowledgements
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-
profit sectors.
Conflicts of interest
The authors have declared that there are no conflicts of interest.
References
Ahankar, H., Ramazani, A., lepokura, K., Lis, T., & Joo, W.S. (2016). Synthesis of pyrrolidinone derivatives
from aniline, an aldehyde and diethyl acetylenedicarboxylate in an ethanolic citric acid solution under
ultrasound irradiation. Green Chemistry, 18 (12), 3582-3593. http://doi.org/10.1039/C6GC00157B.
Andrés-Costa, M.J., Proctor, K., Sabatini, M.T., Gee, A.P., Lewis, S.E., Pico, Y., et al. (2017). Enantioselective
transformation of fluoxetine in water and its ecotoxicological relevance. Scientific Reports, 7(1), 11-13.
http://doi.org/10.1038/s41598-017-15585-1.
Borowiecki, P. (2015). Enantiodifferentiation of promethazine using (S)-(-)-BINOL as the MR chiral solvating
agent: determination of the enantiomeric purity and performance comparison with traditional chiral HPLC.
Tetrahedron: Asymmetry, 26(1), 16-23. https://doi.org/10.1016/j.tetasy.2014.11.018.
Cheng, L., Shen, J., Zhang, Q., Zhang, Y., & Zhang, S. (2015). Simultaneous Determination of Three
Tranquillizers in Lamb Liver by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry.
Food Analytical Methods, 8(7), 1876-82. http://doi.org/10.1007/s12161-014-0069-3.
19

Coles, R., & Kharasch, E. D. (2008). Stereoselective metabolism of bupropion by cytochrome P4502B6
(CYP2B6) and human liver microsomes. Pharmaceutical Research, 25(6): 1405-1411.
https://doi.org/10.1007/s11095-008-9535-1.
Cheng, L., Zhang, Y., Shen, J., Wu, C., & Zhang, S. (2010). GC-MS method for simultaneous determination of
four sedative hypnotic residues in swine tissues. Chromatographia, 71(1), 155-158.
http://doi.org/10.1365/s10337-009-1403-6.
Dodd, S., Burrows, G. D., & Norman, T.R. (2000). Chiral determination of mirtazapine in human blood plasma
by high-performance liquid chromatography. Journal of chromatography. B, Biomedical sciences and
applications, 748(2), 439-43. https://doi.org/10.1016/S0378-4347(00)00353-4.
De Santana, F. J., Jabor, V. A. P., Cesarino, E. J., Lanchote, V. L. & Bonato, P. S. (2010). Enantioselective
analysis of mirtazapine, demethylmirtazapine and 8-hydroxymirtazapine in human urine after solid-phase
microextraction. Journal of Separation Science, 33(2), 268-76. https://doi.org/10.1002/jssc.200900534.
Dayyani, N., Khoee, S., & Ramazani, A. (2015). Design and synthesis of pH-sensitive polyamino-ester
magneto-dendrimers: Surface functional groups effect on viability of human prostate carcinoma cell lines
DU145
.
European
Journal
of
Medicinal
Chemistry,
95,
190-202.
https://doi.org/10.1016/j.ejmech.2015.05.028.
da Fonseca B. M., Moreno, I. E., Barroso, M., Costa, S., Queiroz, J. A., & Gallardo, E. (2013). Determination of
seven selected antipsychotic drugs in human plasma using microextraction in packed sorbent and gas
chromatography-tandem mass spectrometry. Analytical and Bioanalytical Chemistry, 405(12), 3953-3963.
https://doi.org/10.1007/s00216-012-6695-y.
Freynhagen, R., Vogt, J., Lipfert, P., & Muth-Selbacha, U. (2006). Mirtazapine and its enantiomers differentially
modulate acute thermal nociception in rats. Brain Research Bulletin, 69(2), 168-73.
20

https://doi.org/10.1016/j.brainresbull.2005.11.017.
Gocke, E. (1996). Review of the genotoxic properties of chlorpromazine and related phenothiazines. Mutation
Research/Reviews in Genetic Toxicology, 366(1), 9-21. https://doi.org/10.1016/S0165-1110(96)90004-4.
Garmroodi, M., Mohammadi, M., Ramazani, A., Ashjari, M., Mohammadi, J., Sabour, B., et al. (2016).
Covalent binding of hyper-activated rhizomucor miehei lipase (RML) on hetero-functionalized siliceous
supports.
International
Journal
of
Biological
Macromolecules,
86,
208-215.
https://doi.org/10.1016/j.ijbiomac.2016.01.076.
Ganni, B., Kumar, R., Jain, M., Kumar, V.B., Shrivastava, S., & Kumar, P. (2019). Development and validation
of stability indicating HPLC method for the determination of process and degradation related impurities in
telmisartan
drug
substance.
Chemical
Methodologies,
2(3),
145-156.
http://doi.org/10.22034/CHEMM.2018.135684.1063.
He, L., Wang, J., Zhang, G., Liu, R., & Fang, B. (2012). Simultaneous determination of tranquilizers and
carazolol residues in swine tissues by liquid chromatography-tandem mass spectrometry. Analytical Letters,
45 (11) :1377-1389. https://doi.org/10.1080/00032719.2012.675484.
Kirchherr, H., & Kü hnVelten, W. N. (2006). Quantitative determination of forty-eight antidepressants and
antipsychotics in human serum by HPLC tandem mass spectrometry: a multi-level, single-sample approach.
Journal of Chromatography B, 843(1), 100-113. https://doi.org/10.1016/j.jchromb.2006.05.031.
Lauroa, F.V., Franciscob, D.C., Marcelac, R.N., Virginiac, M.A., Elizabethc, M.T., Maria, L.R., et al. (2019).
Design and Synthesis of a diaza-bicyclo-naphthalen-oxiranyl-methanone derivative. Theoretical analysis of
their interaction with cytochrome P450-17A1. Chemical Methodologies, 3(2), 194-210.
https://doi.org/10.22034/chemm.2018.147492.1083.
Jalili-Baleh, L., Babaei, E., Abdpour, S., Foroumadi, A., Ramazani, A., Sharifzadeh, M., et al. (2018). A review
21

on flavonoid-based scaffolds as multi-target-directed ligands (MTDLs) for Alzheimer's disease. European
Journal of Medicinal Chemistry, 152, 570-589. https://doi.org/10.1016/j.ejmech.2018.05.004.
Michishita, T., Franco, P., & Zhang, T. (2010). New approaches of LC-MS compatible method development on
α1-acid glycoprotein-based stationary phase for resolution of enantiomers by HPLC. Journal of Separation
Science, 33(23-24), 3627-37. http://doi.org/10.1002/jssc.201000627.
Malekzadeh, M.A., Ramazani, A., Tabatabaei-Rezaei, S.J., & Niknejad, H. (2017). Design and construction of
multifunctional hyperbranched polymers coated magnetite nanoparticles for both targeting magnetic
resonance imaging and cancer therapy. Journal of Colloid and Interface Science 490, 64-73.
http://doi.org/10.1016/j.jcis.2016.11.014.
Nageswara-Rao, R., & Guru-Prasad, k. (2015). Stereo-specific LC and LC-MS bioassays of antidepressants and
psychotics. Biomedical chromatography, 29(1), 21-40. http://doi.wiley.com/10.1002/bmc.3356.
Rezaei, M., & Ramazani, A. (2019). A novel validated method for the determination of dapoxetine HCl by RP-
HPLC in bulk and tablet dosage forms. Current Pharmaceutical Analysis, 6(14), 622-626.
http://doi.org/10.2174/1573412913666171115162602.
Rezaei, M., Ramazani, A., & Hokmabadi, F. (2018). Quetiapine fumarate syntheses and its determination
methods in the pharmaceutical dosage forms, human plasma and urine by RP-HPLC and other analytical
techniques:
a
review.
Chemical
Methodologies,
2(2),
141-156.
http://doi.org/10.22034/CHEMM.2018.60130.
Rezaei, M., Ramazani, A., & Rouhani, M. (2017). An applicable method for the estimation of tetrabenazine by
simple RP-HPLC in tablet dosage form. Chemical Methodologies, 2(1), 136-144.
http://doi.org/10.22631/CHEMM.2017.104859.1017.
Rosado, T., Oppolzer, D., Cruz, B., Barroso, M., Varela, S., Oliveira, V., et al. (2018). Development and
22

validation of a gas chromatography/tandem mass spectrometry method for simultaneous quantitation of
several antipsychotics in human plasma and oral fluid. Rapid Communication in Mass Spectrometry, 23(32),
2081-2095. https://doi.org/10.1002/rcm.8087.
Stevens, J.C., & Wrighton, S. A. (1993). Interaction of the enantiomers of fluoxetine and norfluoxetine with
human liver cytochrome P450. Journal of Pharmacology and Experimental Therapeutics, 266(2), 964-71.
http://doi.org/10.1016/1056-8719(93)90030-I.
Shalbafan, M., Behbehani, G.R., & Ghasemzadeh, H. (2019). Study of interaction of human serum albumin with
doxorubicin (anti-cancer drug) by docking simulation. Chemical Methodologies, 3(3), 354-359.
https://doi.org/10.22034/chemm.2018.149499.1098.
Vyas, A.J., Nathwani, G.P., Patel, A.I., Patel, N.K., & Patel, A.B. (2019). Validated stability indicating RP-
HPLC DAD method for simultaneous determination of amitriptyline hydrochloride and pregabalin in
presence of stress degradation products in tablet dosage form. Chemical Methodologies, 4(3), 494-508.
http://doi.org/10.22034/CHEMM.2019.154554.1107.
Wang, L., Zhao, H., Qiu, Y., & Zhou, Z. (2006). Determination of four benzodiazepine residues in pork using
multiwalled carbon nanotube solid-phase extraction and gas chromatography–mass spectrometry. Journal
of Chromatography A, 1136(1), 99–105. https://doi.org/10.1016/j.chroma.2006.09.088.
Wong, D. T. , Threlkeld, P. J., & Robertson, D. W. (1991). Affinities of fluoxetine, and its enantiomers, and
other inhibitors of serotonin uptake for subtypes of serotonin receptors. Neuropsychopharmacology :
official publication of the American College of Neuropsychopharmacology, 5(1), 43-47.
https://doi.org/10.1016/0028-3908(91)90128-X.
Yavari, I., & Ramazani, A. (1997). An efficient one-pot synthesis of dialkyl 3H-Naphtho[2,1,b]pyran-2,3-
dicarboxylates mediated by vinyl triphenylphosphonium salt. Synthetic Communications, 27(8), 1385-1390.
23

http://dx.doi.org/10.1080/00397919708006068.
Zhang, J., Shao, B., Yin, J., Wu, Y., & Duan, H. (2009). Simultaneous detection of residues of β-adrenergic
receptor blockers and sedatives in animal tissues by high-performance liquid chromatography/tandem mass
spectrometry.
Journal
of
Chromatography
B,
877(20-21),
1915-22.
http://doi.org/10.1016/j.jchromb.2009.05.025.
Zhao, P.F., Wang, Z.K., Gao, X., Guo, X.J., & Zhao, L.S. (2019). Simultaneous enantioselective determination
of 22 chiral pesticide in fruits and vegetables using chiral liquid chromatography coupled with tandem mass
spectrometry. Food chemistry, 277, 298-306. http://doi.org/10.1016/j.foodchem.2018.10.128.
Zhang, L., Wu, P., Zhang,Y., Jin, Q., Yang, D., Wang, L., & Zhang, J. (2014). A GC/MS method for the
simultaneous determination and quantification of chlorpromazine and diazepam in pork samples. Analytical
methods, 6(2), 503-508. http://doi.org/10.1039/C3AY41130C.
Zhang, L., Wu, P., Jin, Q., Ye, H., Huang, X., & Liu, S. (2016). Simultaneous determination of eight
tranquilizers in pork by ultra-performance liquid chromatography coupled with electrospray tandem mass
spectrometry. Food Analytical Methods 10(2): 354-362. http://doi.org/10.1007/s12161-016-0591-6.
Figure captions
Fig. 1. Chemical structures of the seven chiral antipsychotics.
Fig. 2. MRM chromatograms: (a) control pork sample; (b) 10 ng g-1 of standard spiked pork sample.
Fig. 3 Effect of the extraction solvent (a), sorbent (b) and loading solvent (c) on the recoveries of the seven
antipsychotics (n=3). Experimental conditions: (a) sorbent, C18; loading solvent, methanol-water (5:95, v/v); (b)
extraction solvent, acetonitrile; loading solvent, methanol-water (5:95, v/v); (c) extraction solvent, acetonitrile;
sorbent, C18. Other experimental conditions were indicated as “Materials and Methods” part.
24

25

26

27

Table 1 Retention time, ion pairs, cone voltage and collision energy of MRM for the
optimized LC-MS/MS.
Compound
Precursor ion Retention time MRM transitionsb CV (V)c CE (eV)d
( t1 / t2, min)a
[M+H]+
[M+H]+
[M+H]+
[M+H]+
[M+H]+
[M+H]+
[M+H]+
15.6 / 24.2
19.0 / 23.6
11.5 / 14.4
16.5 / 19.6
9.3 / 10.3
5.2 / 7.0
324.24 > 99.87
324.24 > 57.96
284.57 > 86.04
284.57 > 197.87
309.83 > 147.95
309.83 > 66.93
286.90 > 240.91
286.90 > 268.89
265.94 > 194.96
265.94 > 71.99
239.79 > 183.87
239.79 > 130.94
358.28 > 155.99
358.28 > 341.08
44
18
22
14
22
6
Cyamemazine
Promethazine
Fluoxetine
9
9
18
22
14
26
16
12
24
30
10
5
Oxazepam
35
12
26
Mirtazapine
Bupropion
12.8 / 17.2
Phencynonate
a t1 and t2 means the retention time of the first and second eluted enantiomers.
b The quantification ion transitions are underlined.
c CV means cone voltage;
d CE means collision energy
28

Table 2 Enantioseparation of seven antipsychotics on Chiralpak AGP column with different
organic modifier.
IPA
Acetonitrile
k1
15.07 1.67 4.52 10 1.62 1.67 3.14 15 2.39 1.96 3.70
Methanol
Analytes
%
%
%
k1
α
Rs
α
Rs
k1
α
Rs
Cyamemazine
Promethazine
Fluoxetine
Oxazepam
Mirtazapine
Bupropion
5
5
5
5
5
5
5
15.60 1.10 1.56
12.66 1.11 1.76
14.32 1.50 3.90
5
5
5
2
0
2
7.24 1.37 2.51 10 5.55 1.39 2.78
3.05 1.50 2.62 10 2.96 1.50 2.47
4.0 1.38 2.63 15 2.49 1.55 2.75
5.24 1.00
1.59 1.00
-
-
1.72 1.29 1.53
0.60 1.87 1.59
5
5
1.19 1.32 1.61
0.21 2.57 1.66
11.31 1.41 3.77
6.66 1.64 3.79 15 1.64 1.51 2.23
Phencynonate
Mobile phase: organic modifier/10 mM ammonium acetate buffer (pH 5.8 for IPA, pH 4.5 for
acetonitrile and methanol)
“-”: no separation could be observed under the tested conditions;
Capacity factor of the first eluted enantiomer (k1), separation factor (α) and resolution (Rs).
29